Heart disease is the predominant cause of disability and death in all industrialized nations. Cardiac disease can lead to decreased quality of life and long term hospitalization. In addition, in the United States, it accounts for about 335 deaths per 100,000 individuals (approximately 40% of the total mortality) overshadowing cancer, which follows with 183 deaths per 100,000 individuals. Four categories of heart disease account for about 85-90% of all cardiac-related deaths. These categories are: ischemic heart disease, hypertensive heart disease and pulmonary hypertensive heart disease, valvular disease, and congenital heart disease. Ischemic heart disease, in its various forms, accounts for about 60-75% of all deaths caused by heart disease. In addition, the incidence of heart failure is increasing in the United States. One of the factors that renders ischemic heart disease so devastating is the inability of the cardiac muscle cells to divide and repopulate areas of ischemic heart damage. As a result, cardiac cell loss as a result of injury or disease is irreversible.
Human to human heart transplants have become the most effective form of therapy for severe heart damage. Many transplant centers now have one-year survival rates exceeding 80-90% and five-year survival rates above 70% after cardiac transplantation. Heart transplantation, however, is severely limited by the scarcity of suitable donor organs. In addition to the difficulty in obtaining donor organs, the expense of heart transplantation prohibits its widespread application. Another unsolved problem is graft rejection. Foreign hearts are poorly tolerated by the recipient and are rapidly destroyed by the immune system in the absence of immunosuppressive drugs. While immunosuppressive drugs may be used to prevent rejection, they also block desirable immune responses such as those against bacterial and viral infections, thereby placing the recipient at risk of infection. Infections, hypertension, and renal dysfunction caused by cyclosporin, rapidly progressive coronary atherosclerosis, and immunosuppressant-related cancers have been major complications however.
Cellular transplantation has been the focus of recent research into new means of repairing cardiac tissue after myocardial infarctions. A major problem with transplantation of adult cardiac myocytes is that they do not proliferate in culture. (Yoon et al. (1995) Tex. Heart Inst. J. 22:119). To overcome this problem, attention has focused on the possible use of skeletal myoblasts. Skeletal muscle tissue contains satellite cells which are capable of proliferation. However, methods of purifying and growing these cells are complicated. There is a clear need, therefore, to address the limitations of the current heart transplantation therapies in the treatment of heart disease.
To overcome the limitations of the current heart repair methodologies, the present invention provides isolated muscle cells. In a preferred embodiment, the invention pertains to skeletal myoblasts, compositions including the skeletal myoblasts, and methods for transplanting skeletal myoblasts into subjects. In addition, the invention pertains to cardiomyocytes, methods for inducing the proliferation of cardiomyocytes, and methods for transplanting cardiomyocytes to subjects. The present invention offers numerous advantages over the cells and methods of the prior art.
In one aspect, the invention provides a method for preparing a transplantable muscle cell composition comprising skeletal myoblast cells and fibroblast cells comprising culturing the composition on a surface coated with poly-L-lysine and laminin in a medium comprising EGF such that the transplantable composition is prepared. Preferably, the cells are permitted to double less than about 10 times in vitro prior to transplantation such that the fibroblast to myoblast ratio is approximately 1:2 to 1:1.
In one aspect, the invention provides a transplantable composition comprising skeletal myoblast cells and fibroblast cells and, in one embodiment, can comprise from about 20% to about 70% myoblasts and, preferably, about 40-60% myoblasts or about 50% myoblasts. In another embodiment, the transplantable composition comprises at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 98% or 99% myoblasts.
The muscle cells of the invention may be cultured in vitro prior to transplantation and are preferably cultured on a surface coated with poly-L-lysine and laminin in a medium comprising EGF. Alternatively, the surface can be coated with collagen and the composition cultured in a medium comprising FGF.
The muscle cells of the invention preferably engraft into cardiac tissue after transplantation into a subject. The muscle cells of the invention can endogenously express an angiogenic factor, or can be administered in the form of a composition which comprises an angiogenic factor, or the muscle cells of the invention can be engineered to express an angiogenic gene product in order to induce angiogenesis in the recipient heart.
The invention also provides for modifying, masking, or eliminating an antigen on the surface of a cell in the composition such that upon transplantation of the composition into a subject lysis of the cell is inhibited. In one embodiment, PT85 or W6/32 is used to mask an antigen.
The invention further provides a method for treating a condition in a subject characterized by damage to cardiac tissue comprising transplanting a composition comprising skeletal myoblast cells and fibroblast cells into a subject such that the condition is thereby treated.
The invention further provides a method for treating myocardial ischemic damage comprising transplanting a composition comprising skeletal myoblast cells and, optionally, fibroblast cells into a subject such that the myocardial ischemic damage is thereby treated.
In one embodiment, skeletal myoblast cells of the invention can be induced to become more like cardiac cells. In a preferred embodiment, a cardiac cell phenotype in a skeletal myoblast is promoted by recombinantly expressing a cardiac cell gene product in the myoblast so that the cardiac cell phenotype is promoted. In one embodiment, the gene product is a GATA transcription factor and, preferably is GATA4 or GATA6.